A systematic review and meta-analysis of the sensitivity of antibody tests for the laboratory confirmation of COVID-19

Aim: The aim of this study was to investigate the utility of serological tests for the diagnosis of COVID-19 during the first week of symptom onset in patients confirmed with the real-time RT-PCR. Materials & methods: A systematic review and meta-analysis of 58 publications were performed using data obtained from Academic Search Ultimate, Africa-wide, Scopus, Web of Science and MEDLINE. Results: We found that the highest pooled sensitivities were obtained with ELISA IgM-IgG and chemiluminescence immunoassay IgM tests. Conclusion: Serological tests have low sensitivity within the first week of symptom onset and cannot replace nucleic acid amplification tests. However, serological assays can be used to support nucleic acid amplification tests.

kits, limited adequately equipped laboratory facilities and the need for clinical laboratory scientists are blunting effective response to the pandemic. Consequently, underdiagnosis is resulting in undetected viral transmission making Africa a fertile ground for the emergence of SARS-CoV-2 variants. If diagnostic testing capacity is to be enhanced by using serological assays, then the limitations of serology, specifically demonstration of early antibody in the diagnostic laboratory, need to be defined for accurate interpretation of results.
Serological assays are reliable, simple, and cost-effective techniques that allow direct and indirect detection of infections [5]. Laboratory-based serological methods, such as ELISA, chemiluminescence immunoassay (CLIA) and lateral flow immunochromatographic assay (LFIA) are used as supportive diagnostic tools in the attempt of widening access to diagnosis, screening of asymptomatic persons, and providing information on the immune status of recovered persons to end isolation [6].
Serological tests generally detect IgA, IgG, IgM or total antibodies from patient sera or plasma, that are directed against the SARS-CoV-2 specific spike protein (S) and/or nucleocapsid protein (N). The kinetics of IgA, IgM and IgG antibodies against the specific SARS-CoV-2 proteins informs host immune response and is a critical application of diagnostic tests. Antibody profiling in COVID-19 patients has been described in several studies and it has been shown that an IgM antibody response is detectable as early as 3 days post illness onset and peak levels were observed between the second and the third week, while IgG antibody was detected from day four of illness with peak levels observed between the third to the fourth week [7][8][9][10]. However, after profiling IgM and IgG antibodies in 26 COVID-19 patients in one study, the immunoglobulins either appeared at the same time or varied whether IgM or IgG was detected first [8]. In yet another study, longitudinal profiling of serum, saliva and bronchoalveolar lavage fluid for SARS-CoV-2 specific antibodies, demonstrated the dominance of IgA isotypes within the first week of symptom onset [11]. These observations may highlight the importance of assaying all the three isotypes (IgA, IgM and IgG) in diagnosis of acute COVID-19. Evidence from follow-up studies of COVID-19 patients suggests that serum IgA and IgM antibodies gradually decline after achieving peak levels; while on the contrary IgG persist longer [12,13]. Current data suggest that serological testing can detect infection a few days after the onset of symptoms and might be a suitable approach to complement molecular tests and increase the diagnostic reliability. Additionally, serological tests might be instrumental in low-income countries where access to molecular testing can be difficult.
Previous systematic reviews pooled sensitivity stratified by test type and immunoglobulin class and reported lower sensitivities with the LFIA tests compared with ELISA and CLIA. Hence the use of LFIA tests in the diagnosis of COVID-19 has been questioned. The sensitivity of LFIA, CLIA and ELISA have been reported the lowest during the first week of symptom onset but peaked in the third week or later [14,15]. Even though the current evidence points to an increase in sensitivity later during infection, serological tests still result in many false negatives [14]. The use of serological tests in medical decision making should be accompanied with caution [14]. It is worthy noting that previous reviews included studies mostly from China.
In this systematic review we investigated serological assays and analyzed the results to determine the sensitivity of various serological assays for early detection of antibody response during acute phase of illness to support molecular diagnosis. Our study provides pooled sensitivity stratified by test type, immunoglobulin class and test antigen from studies across the world which included at least 300 samples to assess the performance of serological tests within 7 days of symptom onset.

Data sources
We performed a search on 15 April 2021, using the following databases with no restriction in languages: MEDLINE, Academic Search Ultimate, Africa-Wide Information through EBSCOhost, Web of Science and Scopus. Our search terms were (antibody test or IgG or IgM or IgA) and (diagnostic or RT-PCR) and (SARS-CoV-2 or COVID-19 or coronavirus).

Inclusion & exclusion criteria
Eligible studies were full research articles assessing serological assays as diagnostic tools for the laboratory confirmation of COVID- 19. We included studies in which sensitivity and specificity of COVID-19 serological diagnostic were evaluated against the gold standard RT-PCR as reference. We excluded case reports, review articles, editorials and viewpoints. Research articles with less than 300 samples used to estimate sensitivity and specificity were excluded based on recommendations by Bujang and Adnan [16]  independently screened titles and abstracts, and three (NA Makoah, MM Litabe and M Brink) independently screened full-text papers, and disagreements were resolved by two agreeing.

Data analysis
Four investigators (NA Makoah, T Tipih, M Brink and MM Litabe) extracted and verified data on sensitivity, specificity, serological methods, the immunoglobulin class detected and the antigens targeted.
CMIA, eCLIA and CLIA were all summarized as CLIA because of the similarities in the method.

Statistical analysis
We performed a meta-analysis on pooled studies, by test methods (CLIA, LFIA or ELISA) and test antigen (nucleocapsid [N], receptor-binding domain [RBD], N and spike glycoprotein [S], S, subunit 1 of the spike glycoprotein [S1]), which reported sensitivities and specificities for 7 days and overall. The study aims to identify the best test method and or test antigen type, with the highest pooled sensitivity and specificity results, to diagnose SARS-CoV-2 at 7 day and overall post infection. Statistical analysis was performed using R, version 4.0.2 (R Foundation for Statistical Computing, Vienna, Austria) with 'mada' package [17]. We performed a bivariate metaanalysis by pooling sensitivities and specificities of test method, and for test method and test antigen. For the 7 days meta-analysis, we analyzed pooled sensitivities, while we used both pooled sensitivities and specificities for the overall analysis. Heterogeneity was assessed based level of statistical significance, where p < 0.05 shows that the true effects vary [18]. Results from the meta-analysis were summarized into pooled mean and 95% CI.

Characteristics of the studies
Overall, 2113 records were identified through database searches, and 1389 records were analyzed after duplicate removal. A total of 1062 records were excluded after a full screening of the title and abstract. Finally, 327 full texts were screened, and 58 articles met the inclusion criteria ( Figure 1) . Table 1 summarizes the studies, including countries, test methods and number of samples. The total number of tests extracted exceeds the total number of publications included in the review because more than one method was evaluated in some studies. The maximum number of tests evaluated in a single study was 12 [29].      The global distribution of identified articles include China (n = 14), the USA (n = 10), France (n = 8), Italy (n = 3), Singapore (n = 3), Germany (n = 3), India (n = 3), Taiwan (n = 2), Belgium (n = 2), while Switzerland, Austria, Bahrain, Brazil, Iran, Italy, Japan, Sweden, Thailand, Qatar and the United Kingdom each contributed one article. Two of the selected studies were conducted in more than one country [53,64].
A range of serological assays were evaluated in the identified articles and these include CLIA (n = 64), ELISA (n = 49), LFIA (n = 31), luciferase immunoprecipitation system (n = 3), Immunoblot and flow cytometry-based assays were each reported in single studies. One study used synthetic peptides derived from the open reading frame 1a/b, S and N proteins to develop a chemiluminescent assay to detect IgM and IgG against SARS-CoV-2 [21]. The antigen types described in the articles were N (n = 77), S (n = 28), S1 (n = 25), RBD (n = 15), subunit 2 of the spike glycoprotein (S2 [ n = 8]) and whole virus lysate (n = 4). In four articles, the target antigen was not reported.
Forty-four papers reported diagnostic data on commercial serological kits while 18 studies reported data obtained using in-house developed kits (Table 1). In five publications, in-house assays were evaluated in parallel with commercial assays [23,36,37,42,57].
Sensitivity from onset of symptoms to 7 days We identified 22 articles that reported the sensitivity data from onset of symptoms up to 7 days post symptom onset and extracted data on sensitivity, the antigens targeted, and the antibodies detected (Supplementary Table   10  6). Specificity data based on days after onset of symptoms was not reported in the identified articles. In three articles [59,72,73], sensitivity and specificity data were stratified by days after PCR test and were therefore not included in the analysis. A total of 60 serology methods were extracted including CLIA (n = 26), LFIA (n = 17), ELISA (n = 16) and flow cytometry (n = 1). A total of 61 results were extracted and analyzed since some of the methods gave more than one result for immunoglobulin type detection (Supplementary Table 6). Eight antigen formats were described in the assays and the most frequently evaluated immunoglobulin classes included IgG (n = 36) and IgM (n = 16). Three studies evaluated IgA and reported sensitivities of 37.5 [22], 33.3 [29] and 23% [73]. A Luminex-based assay using the RBD antigen reported a positive percent agreement of 46.15% [33]. We also identified one study [49] reporting a total antibody-based fluorescence enzyme immunoassay system using S1 antigens with 53.9% sensitivity.
We performed a meta-analysis to evaluate the performance of the serological tests within 7 days of post symptoms onset and the results are shown in
The overall performance of serological tests compared with the rRT-PCR was evaluated and the results are shown in Table 4 Table 5). We also evaluated heterogeneity for the LFIA, CLIA and ELISA based methods (Supplementary Table 2). Significant heterogeneity was observed only with the CLIA IgG, ELISA IgG, ELISA IgM-IgG tests.

Discussion
COVID-19 continues to pose a major global healthcare challenge despite the accelerated delivery of vaccination. It remains necessary to identify infection early and isolate the infected individual to reduce the spread of the disease.
In this systematic review and meta-analysis, we investigated the utility of serological assays for the diagnosis of COVID-19 during the first week of symptom development in PCR-positive patients. Studies reporting sensitivity and specificity data based on days after PCR test to categorize days of symptom onset were not incorporated into the analysis. We reasoned that the actual number of days post symptom onset are likely to be underestimated if classified by the number of days after the PCR test. Additionally, we evaluated the overall diagnostic performance of serological test methods for detecting SARS-CoV-2 antibodies in serum from patients with a positive RT-PCR test.
Overall, CLIA IgM-IgG demonstrated superior diagnostic accuracy with sensitivity and specificity of 88.1% (95% CI: 84.26-91.01) and 98.6% (95% CI: 96.34-99.38) respectively. Since the 95% CIs were overlapping, we performed statistical analysis of the pooled mean sensitivities to determine whether they were significantly different across the different sets of tests used. The pooled mean sensitivity obtained with CLIA IgM-IgG was significantly higher than LFIA IgM and CLIA IgG only (Supplementary Table 4). Unlike previous studies that have reported lower sensitivities with the LFIA test method compared with the CLIA and ELISA based assays within each antibody class [14,15,77,78] [14,77,78]. Our results and those from previous studies demonstrate that the clinical utility of serological assays for the detection of SARS-CoV-2 antibodies within 7 days since symptom onset is limited. Patients with 7 days and fewer post symptoms onset could be in a pre-seroconversion state; consequently, higher antibody positivity has been reported at least 14 days post symptoms onset [78][79][80].
We evaluated the immunoglobulin class and antigens that can be targeted to maximize the performance of serological tests. Overall, CLIA based assays measuring the total antibody against the RBD had the best diagnostic accuracy with sensitivity and specificity of 93.3% (95% CI: 88.54-96.03) and 98.8% (95% CI: 96.54-99.59), respectively (Table 5). A previous study reported the highest sensitivity with tests detecting the total antibody against both the N and S antigens [78]. Our search did not yield serological tests detecting the total antibody against the N and S antigens. Within 7 days of developing symptoms, the highest sensitivity was obtained with ELISA IgM-IgG targeting the spike protein, 53.2% (95% CI: 31.52-73.16) ( Table 3). Even though the CLIA total antibody RBD and the ELISA IgM-IgG S had higher estimates of accuracy, the estimated pooled mean sensitivities and specificities were not significantly higher than all the identified test methods targeting other antigens (Supplementary Table 5).
We assessed whether serological tests detecting both IgM and IgG have higher diagnostic sensitivity compared with tests measuring either IgM or IgG. Such knowledge would assist in prioritizing the procurement of serological diagnostic kits. Our study shows that combining IgG and IgM yields higher sensitivity compared with measuring IgM alone with ELISA tests and IgG in LFIA tests within 7 days of symptom onset (Supplementary Table 3). There was no enhanced benefit of assaying both IgM and IgG with the CLIA based assays during the first week of symptom onset. Overall, detecting both IgM and IgG was superior to detecting IgG or IgM with the CLIA and LFIA tests (Supplementary Table 4). However, larger studies would be required to verify these results.
The performance of IgM-based assays was evaluated against the IgG based serological tests in our study. Regarding overall estimates of diagnostic accuracy, CLIA IgM and ELISA IgM serological methods had higher pooled sensitivity estimates compared with the CLIA IgG and ELISA IgG test methods. In LFIA tests, detecting the IgG seemed to be a better choice than assaying the IgM. The meta-analysis by Bastos et al. and Vengesai et al. reported higher pooled sensitivities with the CLIA IgG and LFIA IgG based serological assays compared with the respective IgM test methods albeit with overlapping 95% CIs, while with the ELISA methods higher positivity was obtained by detecting the IgM [14,15]. Regarding sensitivity within 7 days of symptom onset, our findings show that neither IgM nor IgG was a more sensitive marker for COVID-19 diagnosis. That IgM and IgG sensitivities were comparable within the first week of disease onset was expected since both have been detected during the first week of symptom onset [8,81,82]. The early appearance of the IgG may be a consequence of the original antigenic sin effect [83]. IgA has been reported early after infection [84,85] thus is a potential early diagnostic marker for SARS-CoV-2. However, few studies have systematically evaluated IgA in large studies. In our study, we identified only three studies reporting sensitivities of 37.5 [22], 33.3 [29] and 23% [73] within the first week of illness.
This study has some limitations. The magnitude of immune response is influenced by several factors such as age, disease severity and the presence of immunodeficiency disorders which were not considered among the study participants from which blood samples were collected. Studies stratifying the study population according to age, disease severity and immune health are therefore necessary. Studies included in the analysis stratified patients according to the date of symptom onset, relying on participants recalling from memory thus potentially introducing recall bias. In addition, there was insufficient data in the studies to evaluate the cross-reactivity of the serological assays.

Conclusion
Choice of antigen did not appear to influence the outcome of sensitivity although there were differences in sensitivity for different types of assays performed. Our results show that serological tests based on CLIA IgM and ELISA IgM-IgG were the most sensitive during the first week post symptoms onset. As mentioned previously, the role of antibody detection has limitations in acute diagnosis because of the time taken for the development of an endogenous demonstrable antibody response. However, in resource-challenged settings, serology could play a role taking into consideration the limitations of sensitivity when interpreting the results.

Summary points
• The present systematic review presents a synthesis of the sensitivity of antibody tests commonly used to diagnose COVID-19. It also assesses which antibody tests could be used to support RT-PCR for the diagnostic of COVID-19. • Research articles describing or comparing antibody tests for the diagnostic of COVID-19, were extracted from various databases. Those including at least 300 samples tested were included in this study. The sensitivity data were extracted at different time points post positive RT-PCR diagnostic, and a meta-analysis was performed. • The data showed that measuring IgG and IgM yields higher sensitivity compared with measuring IgM alone with ELISA and IgG in lateral flow immunochromatographic assay tests within 7 days of symptom onset and the highest pooled sensitivities were obtained with IgM-IgG and chemiluminescence immunoassay IgM tests within the first 7 days. • Serological tests have low sensitivity within the first week of symptom onset and cannot replace nucleic acid amplification tests. However, serological assays can be used to support nucleic acid amplification tests and have application in surveillance. Serological tests based on chemiluminescence immunoassay IgM and ELISA IgM-IgG were the most sensitive during the first week post symptom onset.

Supplementary data
To view the supplementary data that accompany this paper please visit the journal website at: www.futuremedicine.com/doi/sup pl/10.2217/fvl-2021-0211

Financial & competing interests disclosure
We would like to thank NRF SARChI Vector borne and zoonotic pathogens research (Grant number 98346) and the NRF Thuthuka have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.
No writing assistance was utilized in the production of this manuscript.

Ethical conduct of research
Ethical clearance for the study was obtained from the Health Sciences Research Ethics Committee (HSREC) (ethics no.: UFS-HSD2020/2102/2601) at the University of the Free State.